Novel tumor antigen protein SART-3 and tumor antigen peptides thereof

ABSTRACT

A novel tumor antigen protein and gene therefor, tumor antigen peptides derived from said tumor antigen protein or derivatives thereof as well as medicaments, prophylactics, or diagnostics for tumors using such tumor substances in vitro or in vitro are provided.

TECHNICAL FIELD

The present invention relates to the novel tumor antigen protein, andtumor antigen peptides thereof. More particularly, it relates to thenovel tumor antigen protein and the gene thereof, tumor antigen peptidesderived from the tumor antigen protein, and derivatives of theirsubstances, as well as to medicaments, prophylactics, or diagnostics fortumors that utilize in vivo or in vitro such tumor antigen protein,genes, tumor antigen peptides, or derivatives thereof.

BACKGROUND ART

It is known that immune system, particularly T cells, plays an importantrole in tumor elimination by a living body. Indeed, infiltration oflymphocytes exhibiting cytotoxic effects on tumor cells in human tumorfoci has been observed (Arch Surg., 126:200, 1990), and cytotoxic Tlymphocytes (CTLs) recognizing autologous tumor cells have been isolatedfrom melanomas without great difficulties (e.g., Immunol. Today, 8:385,1987; J. Immunol., 138:989, 1987; and Int. J. Cancer, 52:52, 1992). Inaddition, the results of clinical treatment of melanomas by transfer ofthe CTLs recognizing autologous tumor cells also suggest the importanceof T cells in tumor elimination (J. Natl. Cancer. Inst., 86:1159, 1994).

Although it had long been unknown about target molecules for CTLsattacking autologous tumor cells, the recent advance in immunology andmolecular biology gradually began elucidating such target molecules.Specifically, it has been found that CTL, using the T cell receptors(TCRs), recognizes a complex between a peptide, called tumor antigenpeptide, and a major histocompatibility complex class I antigen (MHCclass I antigen, and in the case of human, referred to as HLA antigen),and thereby attacks autologous tumor cells.

Tumor antigen peptides are generated by degradation of tumor antigenproteins, which are proteins specific for tumors, in cells withproteasomes, which proteins are intracellularly synthesized. The tumorantigen peptides thus generated bind to MHC class I antigens (HLAantigens) in endoplasmic reticulum to form complexes, and the complexesare transported to the cell surface to be presented as an antigen. Atumor-specific CTL recognizes the complex presented as an antigen, andexhibits anti-tumor effects through its cytotoxic action or productionof lymphokines. As a consequence of elucidation of a series of theactions, it has become possible to treat tumors by using tumor antigenproteins or tumor antigen peptides as so-called cancer vaccines toenhance tumor-specific CTLs in the body of a tumor patient.

As a tumor antigen protein, T. Boon et al. identified a protein namedMAGE from human melanoma cells for the first time in 1991 (Science,254:1643, 1991). Subsequently, several additional tumor antigen proteinshave been identified mainly from melanoma cells. Examples of melanomaantigens that have been identified are melanosomal proteins such as amelanocytic tissue-specific protein, gp100 (J. Exp. Med., 179:1005,1994), MART-1 (Proc. Natl. Acad. Sci. USA, 91:3515, 1994), andtyrosinase (J. Exp. Med., 178:489, 1993); MEGE-related proteins that areexpressed not only on melanomas but also on various cancer cells andnormal testicular cells (J. Exp. Med., 179:921, 1994); β-catenin havinga tumor-specific amino acid mutation (J. Exp. Med., 183:1185, 1996); andCDK4 (Science, 269:1281, 1995). Tumor antigen proteins other than thosefrom melanomas have also been identified, including products ofoncogenes such as HER2-neu (J. Exp. Med., 181:2109, 1995) and p53(variant) (Proc. Natl. Acad. Sci. USA, 93:14704, 1996); tumor markerssuch as CEA (J. Natl. Cancer Inst., 87:982, 1995) and PSA (J. Natl.Cancer Inst., 89:293, 1997); and viral proteins such as HPV (J.Immunol., 154:5934, 1995) and EBV (Int. Immunol., 7:653, 1995). Detaileddescriptions of these substances can be found in published reviews (e.g.Immunol. Today, 18:267, 1997; J. Exp. Med., 183:725, 1996; and Curr.Opin. Immunol., 8:628, 1996).

In applications of a tumor antigen protein or a tumor antigen peptide totreatment or diagnosis of tumors, it is important to identify a tumorantigen that can be widely applied to squamous cell carcinomas such asesophageal and lung cancers that occur at a much higher incidencecompared to melanomas. In this relation, the present inventors conductedcloning of a gene encoding a novel tumor antigen protein from squamouscell carcinoma cells derived from esophageal cancer, and identified forthe first time from the tumor cell other than melanomas several tumorantigen peptides that are bound to and presented on HLA antigens ofwhich HLA types are HLA-A24 or HLA-A26 (J. Exp. Med., 187:277, 1998;International Patent Publication WO 97/46676).

When these tumor antigen peptides are clinically applied in practice, itmay be desirable to use two or more different tumor antigen peptidesrather than to use merely one peptide. That is to say, taking intoconsideration the facts that all cancer cells do not express anidentical tumor antigen in common and that two or more different tumorantigen peptides are presented on a single cancer cell, a treatmentusing two or more different tumor antigen peptides is believed to bemore effective. Indeed, in the case of melanoma, development of cocktailformulations comprising two or more peptides has been attempted, since asingle peptide derived from a tumor antigen failed to exhibit adequateeffects (Int J. Cancer, 66:162, 1996; and Int. J. Cancer, 67:54, 1996).Under such circumstances, it is being required to identify novel tumorantigen proteins and tumor antigen peptides that can be widely appliedto squamous cell carcinomas that occur at a higher incidence.

DISCLOSURE OF THE INVENTION

The present invention aims to provide the novel tumor antigen proteinand tumor antigen peptides. Particularly, it aims to provide the noveltumor antigen protein and gene thereof, tumor antigen peptides derivedfrom the tumor antigen protein, and derivatives of their substances, aswell as to medicaments, prophylactics, or diagnostics for tumors thatutilize in vivo or in vitro such tumor antigen protein, genes, tumorantigen peptides, or derivatives thereof. The tumor antigen peptides ofthe present invention include a tumor antigen peptide that is bound toand presented on HLA-A24 that is the HLA antigen carried by about 60% ofthe Japanese people and a tumor antigen peptide that is bound to andpresented on HLA-A2 carried by about 40% of the Japanese and theCaucasians, and, therefore, it can be applied to many patients. Further,the tumor antigen peptides of the present invention may be also appliedto squamous cell carcinomas or the like that is recognized mostfrequently as an etiologic cancer in human, and are expected to haveutilities as novel anti-tumor medicaments. It is known that the squamouscell carcinoma on esophageal or lung cancer among the squamous cellcarcinomas tends to relatively exhibit a resistance to the currentchemotherapy and radiotherapy. In this respect, the development of thetumor antigen peptides of the present invention is desired.

In order to obtain novel tumor antigen protein and tumor antigenpeptides, the present inventors made the following attempts.

First of all, the present inventors prepared a cDNA library fromesophageal cancer cell line KE-4 (FERM BP-5955), and doubly transfectedfibroblast cell line VA-13 (RIKEN CELL BANK, The Institute of Physicaland Chemical Research) with a recombinant plasmid of the library and arecombinant plasmid containing cDNA of HLA-A2402 (one type of HLA-A24).The resulting transfectants were treated with KE-4CTL (FERM BP-5954)that is directed to KE-4, and the amount of produced IFN-γ was measuredto determine whether or not KE-4CTL was activated. As a result of suchextensive screening repeatedly conducted, the present inventors finallysucceeded in cloning one gene encoding a tumor antigen protein althoughwe did not assure that the screening resulted in a novel and usefultumor antigen protein. The inventors named the tumor antigen proteinencoded by the gene “SART-3”. Comparing the base sequence of SART-3 withknown sequences revealed that said base sequence of SART-3 was a novelbase sequence that is different from the KIAA0156 gene registered asAccession No. D63879 at GenBank database in terms of a single base,which function has not been demonstrated.

Further, the present inventors identified tumor antigen peptideportions-residing in the amino acid sequence of SART-3 that are bound toand presented on HLA-A24 and HLA-A2, and demonstrated that such peptideshave activity as a tumor antigen peptide.

The present invention has been completed on the basis of the findings asdescribed above.

Thus, the present invention relates to:

(1) A DNA encoding a protein consisting of an amino acid sequence shownin SEQ ID NO: 1, or a protein variant consisting of an amino acidsequence containing substitution, deletion, and/or addition of one ormore amino acid residues of SEQ ID NO: 1, provided that the protein andthe protein variant give rise to tumor antigen peptides that are capableof binding to an HLA antigen and being recognized by cytotoxic Tlymphocytes;

(2) A DNA consisting of a base sequence shown in SEQ ID NO: 2, or a DNAvariant that hybridizes to the DNA under a stringent condition, providedthat a protein produced and expressed by the DNA or the DNA variantgives rise to tumor antigen peptides that are capable of binding to anHLA antigen and being recognized by cytotoxic T lymphocytes;

(3) An expression plasmid that contains the DNA of the above (1) or (2);

(4) A transformant that is transformed with the expression plasmid ofthe above (3);

(5) A process for producing a recombinant protein, which comprisesculturing the transformant of the above (4), and recovering theexpressed recombinant protein;

(6) A tumor antigen protein that is encoded by the DNA of the above (1)or (2), or is produced by the process of the above (5);

(7) A pharmaceutical composition that comprises as an active ingredientthe DNA of the above (1) or (2), or the protein of the above (6);

(8) A pharmaceutical composition for treating or preventing tumors,which comprises as an active ingredient the DNA of the above (1) or (2),or the protein of the above (6);

(9) A tumor antigen peptide that is a partial peptide derived from theprotein of the above (6), and that is capable of binding to an HLAantigen and being recognized by cytotoxic T lymphocytes, or a derivativethereof having the functionally equivalent properties;

(10) The tumor antigen peptide of the above (9) wherein the HLA antigenis HLA-A24 or HLA-A2, or a derivative thereof having the functionallyequivalent properties;

(11) The tumor antigen peptide of the above (10), which comprises asequence selected from all or part of an amino acid sequence shown inany one of SEQ ID NOs: 3-52, or a derivative thereof having thefunctionally equivalent properties;

(12) The tumor antigen peptide of the above (11), which comprises asequence selected from all or part of an amino acid sequence shown inany one of SEQ ID NOs: 3-9 and 25-29, or a derivative thereof having thefunctionally equivalent properties;

(13) The tumor antigen peptide derivative of the above (11), whichcomprises a sequence selected from all or part of an amino acid sequencewherein the amino acid residue at position 2 and/or the C-terminus inthe amino acid sequence shown in any one of SEQ ID NOs: 3-52 issubstituted by another amino acid residue;

(14) The tumor antigen peptide derivative of the above (13), whichcomprises a sequence selected from all or part of an amino acid sequencewherein the amino acid residue at position 2 and/or the C-terminus inthe amino acid sequence shown in any one of SEQ ID NOs: 3-9 and 25-29 issubstituted by another amino acid residue;

(15) The tumor antigen peptide derivative of the above (13), whichcomprises a sequence selected from all or part of an amino acid sequencewherein the amino acid residue at position 2 in the amino acid sequenceshown in any one of SEQ ID NOs: 3-24 is substituted by tyrosine,phenylalanine, methionine, or tryptophan, and/or the amino acid residueat the C-terminus is substituted by phenylalanine, leucine, isoleucine,tryptophan, or methionine;

(16) The tumor antigen peptide derivative of the above (13), whichcomprises a sequence selected from all or part of an amino acid sequencewherein the amino acid residue at position 2 in the amino acid sequenceshown in any one of SEQ ID NOs: 25-52 is substituted by leucine,methionine, valine, isoleucine, or glutamine, and/or the amino acidresidue at the C-terminus is substituted by valine or leucine;

(17) The tumor antigen peptide derivative of the above (14), whichcomprises a sequence selected from all or part of the amino acidsequence shown in any one of SEQ ID NOs: 53-64;

(18) A pharmaceutical composition for treating or preventing tumors,which comprises as an active ingredient at least one of substancesselected from the tumor antigen peptides and derivatives thereofaccording to any one of the above (9) to (17);

(19) A recombinant DNA comprising at least one of DNAs that encode thetumor antigen peptides or derivatives thereof according to any one ofthe above (9) to (17);

(20) A recombinant polypeptide obtainable by expressing the recombinantDNA of the above (19);

(21) A pharmaceutical composition for treating or preventing tumors,which comprises as an active ingredient the recombinant DNA of the above(19) or the recombinant polypeptide of the above (20);

(22) An antibody that specifically binds to any one of the tumor antigenprotein of the above (6), and the tumor antigen peptide or thederivative thereof according to any one of the above (9) to (17);

(23) An antigen-presenting cell wherein a complex between an HLA antigenand the tumor antigen peptide or the derivative thereof according to anyone of the above (9) to (17) is presented on the surface of a cellhaving antigen-presenting ability, which cell is isolated from a tumorpatient;

(24) An antigen-presenting cell on which a complex between an HLAantigen and a tumor antigen peptide or a derivative thereof ispresented, said antigen-presenting cell being obtainable by allowing acell having antigen-presenting ability isolated from a tumor patient tobe incorporated with the DNA of the above (1) or (2), the tumor antigenprotein of the above (6), the recombinant DNA of the above (19), or therecombinant polypeptide of the above (20);

(25) A pharmaceutical composition for treating tumors, which comprisesas an active ingredient the antigen-presenting cell of the above (23) or(24);

(26) A cytotoxic T lymphocyte that specifically recognizes a complexbetween an HLA antigen and the tumor antigen peptide or derivativethereof according to any one of the above (9) to (17);

(27) A cytotoxic T lymphocyte that specifically recognizes a complexbetween an HLA antigen and a tumor antigen peptide or derivativethereof, which complex is presented on the antigen-presenting cell ofthe above (23) or (24);

(28) A pharmaceutical composition for treating tumors, which comprisesas an active ingredient the cytotoxic T lymphocyte of the above (26) or(27);

(29) A diagnostic agent for tumors, which comprises the tumor antigenpeptide or derivative thereof according to any one of the above (9) to(17), the protein of the above (6), or the recombinant polypeptide ofthe above (20);

(30) Cytotoxic T lymphocyte OK-CTL, of which the deposit number is FERMBP-6818; and

(31) A method for identifying tumor antigen proteins or tumor antigenpeptides, which comprises using OK-CTL of the above (30).

The DNAs of the present invention encode novel tumor antigen proteins,and specific examples of the DNAs include a DNA encoding SART-3 proteinconsisting of an amino acid sequence shown in SEQ ID NO: 1, or a proteinvariant consisting of an amino acid sequence containing substitution,deletion, and/or addition of one or more amino acid residues of theamino acid sequence of SART-3, provided that the protein and the proteinvariant give rise to tumor antigen peptides that are capable of bindingto an HLA antigen and being recognized by cytotoxic T lymphocytes; or aDNA of SART-3 consisting of a base sequence shown in SEQ ID NO: 2, or aDNA variant that hybridizes to the DNA of SART-3 under a stringentcondition, provided that a protein produced and expressed by the DNA andthe DNA variant gives rise to tumor antigen peptides that are capable ofbinding to an HLA antigen and being recognized by cytotoxic Tlymphocytes. The DNA of the present invention is further describedhereinafter following the order established above.

1) DNA Encoding SART-3

“DNA encoding a protein consisting of an amino acid sequence shown inSEQ ID NO: 1” and “a DNA consisting of a base sequence shown in SEQ IDNO: 2” among the DNAs described above refers to a DNA encoding tumorantigen protein SART-3 of the present invention. The DNA may be clonedin accordance with the process described in Examples hereinafter.Further, the cloning of the DNA may be also conducted by, for example,screening a cDNA library derived from cell lines such as esophagealcancer cell line KE-4 (FERM BP-5955) using an appropriate portion of thebase sequence disclosed in GenBank Accession No. D63879 or shown in SEQID NO: 2 in the present specification as a probe for hybridization or aPCR primer. It would be ready for those skilled in the art to achievesuch cloning in accordance with Molecular Cloning 2nd Edt. Cold SpringHarbor Laboratory Press (1989), for example.

2) DNA Encoding a Modified Protein of SART-3 or Allelic Variant Thereof

“DNA encoding a protein variant consisting of an amino acid sequencecontaining substitution, deletion, and/or addition of one or more aminoacid residues of the amino acid sequence of SART-3” among the DNAsdescribed above refers to a DNA that encodes a so-called modifiedprotein, which is artificially prepared, or proteins such as an allelicvariant existing in a living body. The DNA encoding such proteinvariants may be prepared by diverse methods such as site-directedmutagenesis and PCR technique that are described in Molecular Cloning: ALaboratory Manual 2nd Edt. vols. 1-3, Cold Spring Harbor LaboratoryPress (1989). Number of amino acid residue to be substituted, deletedand/or added should be in a range that enables the substitution,deletion, and/or addition in accordance with the well-known methods suchas site-directed mutagenesis as shown above.

3) DNA that Hybridizes to the DNA of SART-3 Under a Stringent Condition

“DNA variant that hybridizes to the DNA of SART-3 under a stringentcondition” among the DNAs described above refers to a DNA thathybridizes to human SART-3 cDNA consisting of the base sequence shown inSEQ ID NO: 2 under a stringent condition, including SART-3 DNAs from allof vertebrate such as rat and mouse, and DNAs encoding a partial proteinof SART-3.

The term “stringent condition” refers to a condition such that ahybridization is conducted in a solution containing 6×SSC (20×SSCrepresents 333 mM Sodium citrate, 333 mM NaCl), 0.5% SDS and 50%formamide at 42° C., and then the hybridized products are washed in asolution of 0.1×SSC, 0.5% SDS at 68° C., or to conditions as describedin Nakayama, et al., Bio-Jikken-Illustrated, vol. 2,“Idenshi-Kaiseki-No-Kiso (A Basis for Gene Analysis)”, pp. 148-151,Shujunsha, 1995.

The DNA variants are cloned by diverse processes such as hybridizationto the DNA shown in SEQ ID NO: 2. Particular procedures for theprocesses such as production of cDNA library, hybridization, selectionof positive colony, and determination of base sequence are well-known,and may be conducted consulting Molecular Cloning as shown above. Probesuseful for the hybridization includes a DNA comprising a base sequencedescribed in SEQ ID NO: 2.

Among the DNAs as described above 1) to 3), a DNA having an ability togenerate a tumor antigen peptide that is capable of binding to an HLAantigen and being recognized by CTLs, and that is derived from a proteinproduced by the expression of the DNA via intracellular degradation,constitutes the DNA encoding tumor antigen protein of the presentinvention, namely, the DNA of the present invention. Particularly, theDNAs of the present invention are those that generate such peptidefragment as a partial peptide consisting of a part of an amino acidsequence of a protein produced by the expression of said DNA, saidpeptide being capable of binding to an HLA antigen, and inducingproduction of cytotoxic actions and cytokines from CTLs specific for thecomplex between the peptide and the HLA antigen that bind to the complexpresenting on the cell surface.

Determination whether or not a candidate DNA may be a DNA encoding atumor antigen protein may be achieved for example by the followingmethod.

An expression plasmid containing a candidate DNA and an expressionplasmid containing a DNA encoding an HLA antigen are doubly transfectedinto fibroblast VA-13 (RIKEN CELL BANK, The Institute of Physical andChemical Research) or COS-7 (ATCC CRL 1651) derived from African greenmonkey kidney. The transfection may be achieved, for example, by theLipofectin method using Lipofectamine reagent (GIBCO BRL). Subsequently,a tumor-responsive CTL that is restricted to the particular HLA antigenused is added to act on the transfectants, and then the amount ofvarious cytokines (for example, IFN-γ) produced by said CTL in responseto the transfectants may be measured, for example, by ELISA to determinewhether or not the candidate DNA is a DNA of the present invention. Inthis context, since SART-3 contains HLA-A24- or HLA-A2-restricted tumorantigen peptide portions, HLA-A24 cDNA (Cancer Res., 55:4248-4252(1995); Genbank Accession No. M64740) and HLA-A2 cDNA (Genbank AccessionNo. M84379) may be used as the above DNA encoding the HLA antigen,whereas those CTLs that are prepared from human peripheral bloodlymphocytes as well as HLA-A24-restricted CTLs such as KE-4CTL (FERMBP-5954) or HLA-A2-restricted CTLs such as OK-CTL (FERM BP-6818) may beused as the above CTL.

The DNA of the present invention as described above can be used as anactive ingredient in a medicament or a pharmaceutical composition. Inaccordance with “pharmaceutical composition” that comprises the DNA ofthe present invention as an active ingredient, administration of the DNAof the present invention to a tumor patient makes treatment orprevention of tumors possible.

By administering a DNA of the present invention incorporated into anexpression vector to a tumor patient according to the following method,the tumor antigen protein is highly expressed in antigen-presentingcells. Tumor antigen peptides that are subsequently generated byintracellular degradation bind to HLA antigen to form complexes, and thecomplexes are densely presented on the antigen-presenting cell surface.As a result, CTLs specific for tumors efficiently proliferate in thebody, and destroy tumor cells. In this way, treatment or prevention oftumors is achieved.

Administration and introduction of the DNA of the present invention intocells may be achieved using viral vectors or according to any one ofother procedures (Nikkei-Science, April, 1994, pp. 20-45; Gekkan-Yakuji,36(1), 23-48 (1994); Jikken-Igaku-Zokan, 12(15), 1994, and referencescited therein).

Examples of the methods using viral vectors include methods in which DNAof the present invention is incorporated into DNA or RNA virus such asretrovirus, adenovirus, adeno-associated virus, herpesvirus, vacciniavirus, poxvirus, poliovirus, or Sindbis virus, and introduced intocells. Among these methods, those using retrovirus, adenovirus,adeno-associated virus, or vaccinia virus are particularly preferred.

Other methods include those in which expression plasmids are directlyinjected intramuscularly (DNA vaccination), liposome method, Lipofectinmethod, microinjection, calcium phosphate method, and electroporation,and DNA vaccination and liposome method is particularly preferred.

In order to allow a DNA of the present invention to act as a medicamentin practice, there are an in vivo method in which DNA is directlyintroduced into the body, and an ex vivo method in which certain cellsare removed from human, and after introducing DNA into said cellsextracorporeally, the cells are reintroduced into the body(Nikkei-Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36(1), 23-48(1994); Jikkenn-Igaku-Zokan, 12(15), 1994; and references citedtherein). An in vivo method is more preferred.

In case of in vivo methods, the DNA may be administered by anyappropriate route depending on the disease and symptoms to be treatedand other factors. For example, it may be administered via intravenous,intraarterial, subcutaneous, intracutaneous, intramuscular route, or thelike. In the case of in vivo methods, the compositions may beadministered in various dosage forms such as solution, and are typicallyformulated, for example, in the form of injection containing DNA of thepresent invention as an active ingredient, to which conventionalcarriers may also be added, if necessary. If a DNA of the presentinvention is included in liposomes or membrane-fused liposomes (such asSendai virus (HVJ)-liposomes), the compositions may be in the form ofliposome formulations such as suspension, frozen drug,centrifugally-concentrated frozen drug, or the like.

Although the amount of a DNA of the present invention in suchformulations may vary depending on the disease to be treated, the ageand the weight of the patient, and the like, it is typical to administer0.0001 mg-100 mg, preferably 0.001 mg-10 mg, of a DNA of the presentinvention every several days to every several months.

In the invention, the term “protein” refers to a protein encoded by thevarious DNAs of the present invention as described above, which has anability as tumor antigen protein to give rise to tumor antigen peptidesvia intracellular degradation that are capable of binding to an HLAantigen and being recognized by CTLs. Specific examples of the proteinsinclude SART-3 comprising an amino acid sequence shown in SEQ ID NO: 1.The proteins of the present invention may be produced in large scaleusing the DNA of the present invention as described above.

Production of tumor antigen proteins by expressing the DNA of thepresent invention may be achieved in accordance with many publicationsand references such as “Molecular Cloning” mentioned above.Particularly, an expression plasmid that replicates and functions inhost cells is constructed by incorporating a DNA of the presentinvention into an appropriate expression vector (e.g., pSV-SPORT1,pCR3). Subsequently, the expression plasmid is introduced intoappropriate host cells to obtain transformants. Examples of host cellsinclude those of prokaryotes such as Escherichia coli, unicellulareukaryotes such as yeast, and cells derived from multicellulareukaryotes such as insects or animals. Gene transfer into host cells maybe achieved by conventional methods such as calcium phosphate method,DEAE-dextran method, electric pulse method, Lipofectin method, or thelike. Desired proteins are produced by culturing the transformants inappropriate medium. The tumor antigen proteins thus obtained may beisolated and purified according to standard biochemical procedures.

It can be demonstrated whether or not a tumor antigen protein of thepresent invention has certain activity by, as described above,expressing the DNA of the present invention within cells to produce theprotein of the present invention, and determining if the peptidefragment generated by intracellular degradation of said protein has theactivity as a tumor antigen peptide. In case of using the tumor antigenprotein as it is, the measurement for the activity can be achieved byallowing the protein to be incorporated into the phagocytes such asmacrophage so as to generate peptide fragments in cells, and thencontacting CTLs to complexes between the peptide fragments and HLAantigen, followed by measuring the amount of various cytokines (forexample, IFN-γ) produced by the CTLs in response to the complexes.

The protein of the present invention as described above can be also usedas an active ingredient in medicament or a pharmaceutical composition.In accordance with “pharmaceutical composition” that comprises theprotein of the present invention as an active ingredient, administrationof the protein of the present invention makes treatment or prevention oftumors possible, for example. When administered to a tumor patient, theprotein of the present invention is introduced into antigen-presentingcells. Tumor antigen peptides that are subsequently generated byintracellular degradation bind to HLA antigen to form complexes, and thecomplexes are presented on the cell surface. CTLs specific for thecomplex efficiently proliferate in the body, and destroy tumor cells. Inthis way, treatment or prevention of tumors is achieved.

Pharmaceutical compositions comprising the tumor antigen protein of thepresent invention may be administered together with an adjuvant in orderto effectively establish the cellular immunity, or may be administeredin a particulate dosage form. For such purpose, those adjuvantsdescribed in the literature (Clin. Microbiol. Rev., 7:277-289, 1994) areapplicable. In addition, liposomal preparations, particulatepreparations in which the ingredient is bound to beads having a diameterof several μm, or preparations in which the ingredient is attached tolipids are also possible. Administration may be achieved, for example,intradermally, hypodermically, or by intravenous injection. Although theamount of a tumor antigen protein of the present invention in suchformulations may vary depending on the disease to be treated, the ageand the weight of the patient, and the like, it is typical to administer0.0001 mg-1000 mg, preferably 0.001 mg-100 mg, more preferably 0.01mg-10 mg of a tumor antigen protein of the present invention everyseveral days to every several months.

In the present invention, the term “tumor antigen peptide” refers to apartial peptide that consists of a part of the tumor antigen protein ofthe present invention and is capable of binding to an HLA antigen andbeing recognized by CTL. Accordingly, any peptide falls within the scopeof the tumor antigen peptide of the present invention, regardless of itslength or its position in the amino acid sequence of the presentprotein, as long as the peptide consists of a part of the amino acidsequence of the present protein and a complex between said peptide andan HLA antigen is capable of being recognized by CTL. Such tumor antigenpeptides of the present invention can be identified by synthesizing acandidate peptide which consists of a part of the tumor antigen proteinof the present invention and conducting an assay for determining whetheror not a complex between the candidate peptide and an HLA antigen isrecognized by CTL, in other words, whether or not the candidate peptidehas the activity as a tumor antigen peptide.

In this connection, synthesis of peptides may be conducted according toa method usually used in peptide chemistry. Examples of such knownmethods are those described in the literatures including “PeptideSynthesis”, Interscience, New York, 1966; “The Proteins”, vol. 2,Academic Press Inc., New York, 1976; “Pepuchido-Gosei”, Maruzen Co.Ltd., 1975; “Pepuchido-Gosei-no-Kiso-to-Jikkenn”, Maruzen Co. Ltd.,1985; and “Iyakuhin-no-Kaihatu, Zoku, vol. 14, Peputido-Gosei”, HirokawaShoten, 1991.

Next, methods for identifying tumor antigen peptides of the presentinvention are further described below.

The respective sequence rules (motifs) of antigen peptides that arebound to and presented on the following HLA types have been known; HLA-A1, -A0201, -A0204, -A0205, -A0206, -A0207, -A11, -A24, -A31, -A6801,-B7, -B8, -B2705, -B37, -Cw0401, and -Cw0602 (see, e.g., Immunogenetics,41:178, 1995). Regarding the motif for HLA-A24, for example, it is knownthat in the sequence of peptides consisting of 8 to 11 amino acids, theamino acid at position 2 is tyrosine, phenylalanine, methionine, ortryptophan, and the amino acid at the C-terminus is phenylalanine,leucine, isoleucine, tryptophan, or methionine (J. Immunol., 152:3913,1994; Immunogenetics, 41:178, 1995; J. Immunol., 155:4307, 1994).Likewise, the motifs shown in the following Table 1 are known for HLA-A2(Immunogenetics, 41:178, 1995; J. Immunol., 155:4749, 1995). TABLE 1Amino acid at the second Type of HLA-A2 position from N-terminus Aminoacid at C-terminus HLA-A0201 L, M V, L HLA-A0204 L L HLA-A0205 V, L, I,M L HLA-A0206 V, Q V, L HLA-A0207 L L(the peptides are 8-11 amino acids in length)

In addition, any peptide sequence expected to be capable of binding toHLA antigens may be searched on internet using a software of NIH BIMAS(http://bimas.dcrt.nih.gov/molbio/hla_bind/).

By analysis of antigen peptides bound to various HLA molecules, it hasbeen shown that the length of the peptides is usually about 8 to 14amino acids long, although antigen peptides of 14 or more amino acids inlength are also observed for HLA-DR, -DP, and -DQ (Immunogenetics,41:178, 1995).

It is easy to select peptide portions involved in such motifs from theamino acid sequence of the protein of the present invention. Suchpeptide portions involved in the above motif structures can be easilyselected by inspecting the amino acid sequence of tumor antigen proteinSART-3 (SEQ ID NO: 1). Further, it is easy to select any sequenceexpected to be capable of binding to HLA antigens by search on internetas shown above. Tumor antigen peptides of the present invention can beidentified by synthesizing candidate peptides thus selected according tothe method described above and conducting an assay for determiningwhether or not a complex between the candidate peptide and an HLAantigen is recognized by CTL, in other words, whether or not a candidatepeptide has an activity as a tumor antigen peptide.

A specific example of method for identifying tumor antigen peptides ofthe present invention is a method described in J. Immunol., 154:2257,1995. Specifically, peripheral blood lymphocytes are isolated from ahuman who is positive for the type of an HLA antigen that is expected topresent the candidate peptide, and are stimulated in vitro by adding thecandidate peptide. If the candidate induces CTL that specificallyrecognizes the HLA-antigen-presenting cells pulsed with the candidatepeptide, it is indicated that the particular candidate peptide mayfunction as a tumor antigen peptide. In this connection, the presence orabsence of CTL induction can be detected, for example, by measuring theamount of various cytokines (for example, IFN-γ) produced by CTLs inresponse to the antigen peptide-presenting cells using, for example, anELISA method. Alternatively, a method in which the cytotoxicity of CTLsagainst antigen peptide-presenting cells labeled with ⁵¹Cr is measured(⁵¹Cr release assay, Int. J. Cancer, 58:317, 1994) may also be used forsuch detection.

Furthermore, the above detection can also be achieved as follows. Anexpression plasmid expressing a cDNA for the type of an HLA antigen thatis expected to present the candidate peptide is incorporated into, forexample, COS-7 cells (ATCC No. CRL1651) or VA-13 cells (RIKEN CELL BANK,The Institute of Physical and Chemical Research), and the resultantcells are pulsed with the candidate peptide. The cells are then reactedwith the CTLs that are restricted to the type of the HLA antigenexpected to present the candidate peptide as described above, and theamount of various cytokines (for example, IFN-γ) produced by said CTLsis measured (J. Exp. Med., 187:277, 1998).

SART-3 contains HLA-A24- or HLA-A2-restricted tumor antigen peptideportions. In order to identify HLA-A24-restricted tumor antigenpeptides, HLA-A24 cDNA (Cancer Res., 55:4248-4252, 1995, GenbankAccession No. M64740) can be used as a cDNA encoding the HLA antigen,whereas those CTLs such as KE-4CTL (FERM BP-5954) as well as CTLs thatare prepared by peptide-stimulation of human peripheral bloodlymphocytes can be used as the CTLs described above. Likewise, forHLA-A2-restricted tumor antigen peptides, identification of such tumorantigen peptides can be achieved using HLA-A2 cDNA (Genbank AccessionNo. M84379), and using as the CTLs described above those CTLs such asOK-CTL (FERM BP-6818) as well as CTLs that are prepared bypeptide-stimulation of human peripheral blood lymphocytes.

Specific examples of various assays as described above are illustratedin Examples 4, 6, and 8 hereinafter.

In cases like HLA-A26 wherein a relevant peptide motif is notelucidated, tumor antigen peptides of the present invention can beidentified, for example, according to the method described in WO97/46676, which method is different from that in the above cases whereinthe sequence rules (motifs) have been elucidated, provided that a CTLline recognizing a complex between HLA-A26 and a tumor antigen peptideis available.

The methods for identifying tumor antigen peptides as described abovemay be hereinafter collectively referred to as “assay methods for tumorantigen peptides”.

As described above, it is known that the sequences of tumor antigenpeptides that are bound to and presented on HLA-A24 obey a certain rule(motif), and in particular, the motif is that, in a sequence of apeptide consisting of 8 to 11 amino acids, the amino acid at position 2is tyrosine, phenylalanine, methionine, or tryptophan, and the aminoacid at the C-terminus is phenylalanine, leucine, isoleucine,tryptophan, or methionine (J. Immunol., 152:3913, 1994; Immunogenetics,41:p178, 1995; J. Immunol., 155:p4307, 1994). Likewise, a similar rule(motif) can be found in the sequences of tumor antigen peptides that arebound to and presented on HLA-A2, and in particular, the motifs shown inthe above Table 1 are known (Immunogenetics, 41, p178, 1995; J.Immunol., 155:p4749, 1995). As shown above, sequences expected to becapable of binding to HLA antigens may be further searched on internetusing NIH BIMAS software (http://bimas.dcrt. nih.gov/molbio/hla_bind/).

Accordingly, HLA-A24- and HLA-A2-restricted tumor antigen peptides amongthe tumor antigen peptides of the present invention are exemplified bythose tumor antigen peptides that are partial peptides involved in suchmotif structures or structures expected to be capable of binding to theHLAs in the amino acid sequence of SART-3 shown in SEQ ID NO: 1 and thatare capable of binding to respective HLA antigens and being recognizedby CTLs.

Particular examples of HLA-A24-restricted tumor antigen peptidesdescribed above include those tumor antigen peptides that comprise allor part of an amino acid sequence shown in any one of SEQ ID NOs: 3-24and that are capable of binding to an HLA-A24 antigen and beingrecognized by CTL. Likewise, particular examples of HLA-A2-restrictedtumor antigen peptides include those tumor antigen peptides thatcomprise all or part of an amino acid sequence shown in any one of SEQID NOs: 25-52 and that are capable of binding to an HLA-A2 antigen andbeing recognized by CTL.

Specifically, examples of tumor antigen peptides of the presentinvention include:

1) peptides that consist of an amino acid sequence shown in any one ofSEQ ID NOs: 3-52, and

2) peptides that comprise the full length or a consecutive portion of anamino acid sequence shown in any one of SEQ ID NOs: 3-52 and that areelongated in the N-terminal and/or C-terminal direction as compared tosaid amino acid sequence, or peptides consisting of a consecutiveportion of an amino acid sequence shown in any one of SEQ ID NOs: 3-52,

said peptides being capable of binding to respective HLA antigens andbeing recognized by CTLs. The peptides in the above 2) may be about 8-11amino acids in length in view of the fact that they are bound andpresented by respective HLA antigens.

Suitable examples of HLA-A24-restricted tumor antigen peptides of thepresent invention include those tumor antigen peptides that comprise allor part of the amino acid sequence shown in any one of SEQ ID NOs: 3-9and that are capable of binding to an HLA-A24 antigen and beingrecognized by CTL. Specifically, examples are:

1) peptides that consist of the amino acid sequence shown in any one ofSEQ ID NOs: 3-9, and

2) peptides that comprise the full length or a consecutive portion ofthe amino acid sequence shown in any one of SEQ ID NOs: 3-9 and that areelongated in the N-terminal and/or C-terminal direction as compared tosaid amino acid sequence, or peptides that consist of a consecutiveportion of the amino acid sequence shown in any one of SEQ ID NOs: 3-9,

said peptides being capable of binding to HLA-A24 antigens and beingrecognized by CTLs. The peptides in the above 2) may be about 8-11 aminoacids in length in view of the fact that they are bound to and presentedon HLA-A24 antigens.

Suitable examples of HLA-A2-restricted tumor antigen peptides of thepresent invention include those tumor antigen peptides that comprise allor part of the amino acid sequence shown in any one of SEQ ID NOs: 25-29and that are capable of binding to an HLA-A2 antigen and beingrecognized by CTL. Specifically, examples are:

1) peptides that consist of the amino acid sequence shown in any one ofSEQ ID NOs: 25-29, and

2) peptides that comprise the full length or a consecutive portion ofthe amino acid sequence shown in any one of SEQ ID NOs: 25-29 and thatare elongated in the N-terminal and/or C-terminal direction as comparedto said amino acid sequence, or peptides that consist of a consecutiveportion of the amino acid sequence shown in any one of SEQ ID NOs:25-29,

said peptides being capable of binding to HLA-A2 antigens and beingrecognized by CTLs. The peptides in the above 2) may be about 8-11 aminoacids in length in view of the fact that they are bound to and presentedon HLA-A2 antigens.

In the present invention, the term “derivative having propertiesfunctionally equivalent to those of a tumor antigen peptide”(hereinafter may be simply referred to as tumor antigen peptidederivative) refers to an altered peptide, of which the amino acidsequence contains alteration of one or more, preferably one to several,amino acid residues of an amino acid sequence of a tumor antigen peptideof the present invention, and which has the properties as a tumorantigen peptide, that are to be capable of binding to an HLA antigen andbeing recognized by CTL. Accordingly, all altered peptides fall withinthe scope of tumor antigen peptide of the present invention so long asthey contains alteration of one or more amino acid residues of an aminoacid sequence of a tumor antigen peptide of the present invention, andhave the properties as tumor antigen peptides, that is, are capable ofbinding to HLA antigens and being recognized by CTLs.

In this context, “alteration” of an amino acid residue meanssubstitution, deletion and/or addition (including addition of aminoacids to the N-terminus and/or the C-terminus of the peptide) of anamino acid residue, with substitution of an amino acid residue beingpreferred. For alterations involving substitution of an amino acidresidue, although the number and the position of amino acid residues tobe substituted may be determined arbitrarily so long as the activity asa tumor antigen peptide is retained, it is preferred that one to severalresidues are substituted in light of the fact that tumor antigenpeptides are usually about 8 to 14 amino acids in length as describedabove.

A preferred length of tumor antigen peptide derivatives of the presentinvention is about 8 to 14 amino acids as in case of the tumor antigenpeptide described above, although derivatives of 14 or more amino acidslong may also be possible for HLA-DR, -DP, and -DQ.

Such tumor antigen peptide derivatives of the present invention may beidentified by synthesizing altered peptides that contain alteration of apart of a tumor antigen peptide of the present invention in accordancewith the above preparation of peptide, and by conducting the above assayfor tumor antigen peptides.

As described above, the sequence rules (motifs) for peptides that arebound to and presented on HLA types such as HLA-A1, -A0201, -A0204,-A0205, -A0206, -A0207, -A11, -A24, -A31, -A6801, -B7, -B8, -B2705,-B37, -Cw0401, and -Cw0602 have been elucidated. As shown above, peptidesequences expected to be capable of binding to HLA antigens may befurther searched on internet (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Consequently, tumor antigen peptide derivativescontaining the alteration of the amino acids in a tumor antigen peptideof the present invention can be prepared on the basis of such motifs.

For example, regarding the motif for antigen peptides that are bound toand presented on HLA-A24, it is known as described above that in thesequence of a peptide consisting of 8 to 11 amino acids, the amino acidat position 2 is tyrosine, phenylalanine, methionine, or tryptophan, andthe amino acid at the C-terminus is phenylalanine, leucine, isoleucine,tryptophan, or methionine (J. Immunol., 152:3913, 1994; Immunogenetics,41:178, 1995; J. Immunol., 155:4307, 1994). Likewise, the motifs shownin the above Table 1 are known for HLA-A2. In addition, peptidesequences expected to be capable of binding to HLA antigens is laid openon internet (http://bimas.dcrt.nih.gov/molbio/hla_bind/), and amino acidresidues having properties similar to those of amino acids according tothe motifs may also be possible. Accordingly, examples of tumor antigenpeptide derivatives of the present invention include those peptidederivatives that comprise all or part of an amino acid sequence of thetumor antigen peptide of the present invention in which one or moreamino acid residues at any positions that may be allowed forsubstitution according to the motifs (for HLA-A24 and HLA-A2, position 2and the C-terminus) are substituted by other amino acids (preferably,which is the amino acid expected to be capable of binding to theantigens according to the above internet), and which derivatives haveactivity of binding to HLA antigens and being recognized by CTLs.Preferred examples are those tumor antigen peptide derivatives thatcomprise all or part of an amino acid sequence in which amino acidresidues to be substituted are selected from those at said positionsaccording to the above motifs, and which derivatives have the aboveactivity. A preferred length of “all or part” of an amino acid sequenceis about 8 to 14 amino acids, although it may be a length of 14 or moreamino acids for HLA-DR, -DP, and -DQ.

Examples of HLA-A24- or HLA-A2-restricted tumor antigen peptidederivatives include those peptide derivatives that comprise all or partof an amino acid sequence in which one or more amino acid residues atpositions that are allowed for substitution according to the abovemotifs, specifically, at position 2 and/or the C-terminus, of a peptidederived from the amino acid sequence of SART-3 having a binding motiffor HLA-A24 or HLA-A2 are substituted by other amino acid residues(preferably, which is the amino acid expected to be capable of bindingto the antigens according to the above internet), and which derivativeshave the above activity. Preferred examples are those tumor antigenpeptide derivatives that comprise all or part of an amino acid sequencein which the amino acid residues at position 2 and/or the C-terminus aresubstituted by the amino acid residues involved according to the abovemotifs, and which derivatives have the above activity. In such HLA-A24-or HLA-A2-restricteid tumor antigen peptide derivatives, a preferredlength of “all or part” of the amino acid sequence is about 8 to 11amino acids.

In particular, examples are those tumor antigen peptide derivatives thatcomprise all or part of an amino acid sequence in which the amino acidresidues at position 2 and/or the C-terminus of an amino acid sequenceshown in any one of SEQ ID NOs: 3 to 52 are substituted by other aminoacid residues (preferably, which is the amino acid expected to becapable of binding to the antigens according to internet as shown above)and which derivatives have the above activity. Preferred examples arethose tumor antigen peptide derivatives that comprise all or part of anamino acid sequence in which the amino acid residues at position 2and/or the C-terminus of an amino acid sequence shown in any one of SEQID NOs: 3 to 52 are substituted by the amino acid residues involvedaccording to the above motifs and which derivatives have the aboveactivity. Specifically, examples of HLA-A24-restricted tumor antigenderivatives are those tumor antigen peptide derivatives that compriseall or part of an amino acid sequence in which the amino acid residue atposition 2 of an amino acid sequence shown in any one of SEQ ID NOs: 3to 24 is substituted by tyrosine, phenylalanine, methionine, ortryptophan and/or the amino acid residue at the C-terminus issubstituted by phenylalanine, leucine, isoleucine, tryptophan, ormethionine and which derivatives have the above activity. Likewise,examples of HLA-A2-restricted tumor antigen derivatives are those tumorantigen peptide derivatives that comprise all or part of an amino acidsequence in which the amino acid residue at position 2 of an amino acidsequence shown in any one of SEQ ID NOs: 25 to 52 is substituted byleucine, methionine, valine, isoleucine, or glutamine, and/or the aminoacid residue at the C-terminus is substituted by valine or leucine andwhich derivatives have the above activity.

Suitable examples of HLA-A24-restricted tumor antigen peptidederivatives of the present invention are those tumor antigen peptidederivatives that comprise all or part of an amino acid sequence in whichthe amino acid residues at position 2 and/or the C-terminus of the aminoacid sequence shown in any one of SEQ ID NOs: 3 to 9 are substituted byother amino acid residues and which derivatives have the above activity.More preferred examples are those tumor antigen peptide derivativescomprise all or part of an amino acid sequence in which the amino acidresidue at position 2 of an amino acid sequence shown in any one of SEQID NOs: 3 to 9 is substituted by tyrosine, phenylalanine, methionine, ortryptophan and/or the amino acid residue at the C-terminus issubstituted by phenylalanine, leucine, isoleucine, tryptophan, ormethionine and which derivatives have the above activity. Suitableexamples of such tumor antigen peptide derivatives are shown in SEQ IDNOs: 53 to 59.

Suitable examples of HLA-A2-restricted tumor antigen peptide derivativesof the present invention are those tumor antigen peptide derivativesthat comprise all or part of an amino acid sequence in which the aminoacid residues at position 2 and/or the C-terminus of the amino acidsequence shown in any one of SEQ ID NOs: 25 to 29 are substituted byother amino acid residues and which derivatives have the above activity.More preferred examples are those tumor antigen peptide derivativescomprise all or part of an amino acid sequence in which the amino acidresidue at position 2 of an amino acid sequence shown in any one of SEQID NOs: 25 to 29 is substituted by leucine, methionine, valine,isoleucine, or glutamine, and/or the amino acid residue at theC-terminus is substituted by valine or leucine, and which derivativeshave the above activity. Suitable examples of such tumor antigen peptidederivatives are shown in SEQ ID NOs: 60 to 64.

A tumor antigen peptide or its derivative of the present invention canbe used solely or together with other one or more of them as apharmaceutical composition for treating or preventing tumors. Namely,the present invention provides a pharmaceutical composition fortreatment or prevention for tumors, which comprises the tumor antigenpeptides or derivatives thereof as an active ingredient. When thecomposition for treating or preventing tumors which comprises as anactive ingredient a tumor antigen peptide or its derivative of thepresent invention is administered to a SART-3-positive patient, thetumor antigen peptide or derivative thereof is presented with an HLAantigen of antigen-presenting cells, and therefore, CTLs specific forthe presented HLA antigen complex proliferates and destroys the tumorcells. As a result, the tumor of the patient may be treated, orproliferation or metastasis of the tumor may be prevented. SART-3 isdeveloped extensively on the squamous cell carcinoma such as esophagealcancer, and therefore, the composition for treating or preventing tumorsaccording to the present invention is advantageous in terms of wideapplicability. The squamous cell carcinoma often exhibits a resistanceto chemotherapy and radiotherapy, and, therefore, the composition fortreating tumors of the present invention also can achieve an increasedtherapeutic effect by its combined use.

The composition for treating or preventing tumors comprising as anactive ingredient a tumor antigen peptide or its derivative of thepresent invention may be administered together with an adjuvant in orderto effectively establish the cellular immunity, or may be administeredin a particulate dosage form. For such purpose, those adjuvantsdescribed in the literature (Clin. Microbiol. Rev., 7:277-289, 1994) areapplicable. In addition, liposomal preparations, particulatepreparations in which the ingredient is bound to beads having a diameterof several μm, or preparations in which the ingredient is attached tolipids are also possible. Administration may be achieved, for example,intradermally, hypodermically, or by intravenous injection. Although theamount of a tumor antigen peptide or its derivative of the presentinvention in the formulation to be administered may be adjusted asappropriate depending on, for example, the disease to be treated, theage and the body weight of the particular patient, it is typical toadminister 0.0001 mg to 1000 mg, preferably 0.001 mg to 100 mg, and morepreferably 0.01 mg to 10 mg every several days to every several months.

Furthermore, a recombinant DNA that contains at least one DNA encoding atumor antigen peptide or its derivative of the present invention, or arecombinant polypeptide obtainable by expression of said recombinant DNAmay be also comprised as an active ingredient in the composition fortreating or preventing tumors according to the present invention, whichdetails are provided below.

In this connection, the term “recombinant DNA” refers to any DNAencoding a partial polypeptide, a partial peptide consisting of a partof the tumor antigen protein of the present invention, derivativesthereof, polytope in which such peptides are combined, or the like. AllDNAs fall within the scope of recombinant DNA of the present inventionso long as they contain at least one DNA encoding the tumor antigenpeptide or its derivative of the present invention. Such recombinant DNAmay be incorporated into a suitable expression vector to make an activeingredient comprised in the pharmaceutical composition for treating orpreventing tumors.

The term “polytope” refers to a combined peptide of many CTL epitopes,and DNAs encoding such polytopes have recently been used for DNAvaccination. See, for example, J. of Immunology, 160, p 1717, 1998. DNAencoding the polytope of the present invention can be prepared byligating one or more DNAs encoding the tumor antigen peptide or itsderivative of the present invention each other, and, if desired,ligating a DNA encoding other tumor antigen peptide(s).

Recombinant DNA of the present invention can be easily preparedaccording to typical DNA synthesis and genetic engineering method, forexample, according to the description of a standard text such as“Molecular Cloning”, 2nd ed., Cold Spring Harbor Laboratory Press(1989). Incorporation of such recombinant DNA into expression vectorsmay be also conducted according to the standard text and the like.

Determination whether or not a recombinant DNA of the present inventionas prepared above may generate tumor antigen peptides that are capableof binding to HLA antigens and being recognized by CTLs may be achievedin accordance with, for example, the method as mentioned above fordetermining the activity of DNA of the present invention. Likewise, amethod for using the present recombinant DNA as medicaments orprophylactics may be in accordance with the method for the DNA of thepresent invention.

As shown above, “recombinant polypeptide” obtainable by expression ofthe recombinant DNA of the invention may also be used for apharmaceutical composition for treating or preventing tumors.

The recombinant polypeptide of the invention may be prepared in asimilar manner to that for the protein of the invention as describedabove. Likewise, determination whether or not a recombinant polypeptideof the present invention as prepared above may have certain activity maybe achieved in accordance with a similar manner to that for the proteinof the present invention. Further, a method for using the presentrecombinant polypeptide as medicaments or prophylactics may be inaccordance with the above method for the protein or peptide of thepresent invention.

The present invention also provides antibodies that specifically bind toa protein of the present invention, a tumor antigen peptide of thepresent invention or a derivative thereof. Such antibodies are easilyprepared, for example, according to a method described in “Antibodies: ALaboratory Manual”, Lane, H. D. et al. eds., Cold Spring HarborLaboratory Press, New York, 1989. Specifically, antibodies thatrecognize a tumor antigen peptide or its derivative of the presentinvention and antibodies that further neutralize its activity may easilybe prepared using the tumor antigen peptide or derivative thereof toappropriately immunize an animal in the usual manner. Such antibodiesmay be used in affinity chromatography, immunological diagnosis, and thelike. Immunological diagnosis may be selected as appropriate fromimmunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), a fluorescent or luminescent assay, and the like.

A tumor antigen peptide, derivative thereof, tumor antigen protein, genetherefor of the present invention, or a recombinant DNA or recombinantpolypeptide of the present invention may also be used in vitro fortreatment of tumor patients as follows.

On usage of a tumor antigen peptide, derivative thereof, tumor antigenprotein, or gene therefor in treatment of tumors, it is important toestablish an administration method which can efficiently induce specificCTLs in the body of a patient. As one of the means therefor, the presentinvention provides an antigen-presenting cell in which a complex betweenan HLA antigen and a tumor antigen peptide or its derivative of thepresent invention is presented on the surface of a cell havingantigen-presenting ability isolated from a tumor patient, and alsoprovides a pharmaceutical composition for treating tumors, whichcomprises said antigen-presenting cell as an active ingredient.

In this context, the “cell having antigen-presenting ability” is notlimited to a specific cell so long as it is a cell expressing on itscell surface an HLA antigen allowing a tumor antigen peptide or itsderivative of the present invention to be presented, and dendriticcells, which is reported to have especially a high antigen-presentingability, are preferred.

Substances to be added to prepare an antigen-presenting cell of thepresent invention from the above-mentioned cell having anantigen-presenting ability may be tumor antigen peptides or theirderivatives of the present invention, as well as DNAs, proteins,recombinant DNAs or recombinant polypeptides of the present invention.When used in the form of a protein or DNA, it is necessarily introducedinto cells.

In order to prepare antigen-presenting cells of the present invention,cells having an antigen-presenting ability are isolated from a tumorpatient, and pulsed ex vivo with a tumor antigen peptide, a derivativethereof, a tumor antigen protein, or recombinant polypeptide of thepresent invention to present a complex between an HLA antigen and saidtumor antigen peptide or derivative thereof (Cancer Immunol.Immunother., 46:82, 1998; J. Immunol. 158:p 1796, 1997; Cancer Res.,59:1184, 1999). When dendritic cells are used, antigen-presenting cellsof the present invention may be prepared, for example, by isolatinglymphocytes from peripheral blood of a tumor patient using Ficollmethod, removing non-adherent cells, incubating the adherent cells inthe presence of GM-CSF and IL-4 to induce dendritic cells, andincubating and pulsing said dendritic cells with a tumor antigen peptideor tumor antigen protein of the present invention, or the like.

When antigen-presenting cells of the present invention are prepared byintroducing a DNA or a recombinant DNA of the present invention into theaforementioned cells having an antigen-presenting ability, said gene maybe in the form of DNA or RNA. In particular, DNA may be used consulting,for example, Cancer Res., 56:5672, 1996 or J. Immunol., 161:p5607, 1998,and RNA may be used by consulting, for example, J. Exp. Med., 184:p 465,1996.

A pharmaceutical composition for treating tumors which comprises theabove antigen-presenting cells as an active ingredient preferablycontains physiological saline, phosphate buffered saline (PBS), medium,or the like to stably maintain the antigen-presenting cells. It may beadministered, for example, intravenously, subcutaneously, orintradermally. By reintroducing such composition for treating tumorswhich comprises antigen-presenting cells as an active ingredient intothe body of the patient, specific CTLs are efficiently induced inSART-3-positive patient so as to achieve treatment of the tumor. Itshould be undisputed that the HLA types need be compatible between thepatient and the peptide used, such that an HLA-A24-restricted tumorantigen peptide or a derivative thereof must be used with anHLA-A24-positive tumor patient.

In addition, in vitro use of a tumor antigen peptide, a derivativethereof, a tumor antigen protein, a DNA therefor a recombinant DNA orrecombinant polypeptide according to the present invention in thefollowing adoptive immunotherapy may be provided as another example oftheir use.

For melanomas, it has been observed that an adoptive immunotherapywherein tumor-infiltrating T cells taken from the patienthimself/herself are cultured ex vivo in large quantities, and thenreturned into the patient, achieves a therapeutic effect (J. Natl.Cancer. Inst., 86:1159, 1994). Likewise, in mouse melanoma, suppressionof metastasis has been observed by in vitro stimulation of splenocyteswith tumor antigen peptide TRP-2, thereby proliferating CTLs specificfor the tumor antigen peptide, and administering said CTLs into amelanoma-grafted mouse (J. Exp. Med., 185:453, 1997). This resulted fromin vitro proliferation of CTLs that specifically recognize the complexbetween an HLA antigen of antigen-presenting cells and the tumor antigenpeptide. Accordingly, a method for treating tumors is believed to beuseful, which comprises stimulating in vitro peripheral bloodlymphocytes from a patient using a tumor antigen peptide, a derivativethereof, a tumor antigen protein, or a DNA therefor according to thepresent invention to proliferate tumor-specific CTLs, and subsequentlyreturning the CTLs into the patient.

Thus, the present invention provides CTLs that specifically recognize acomplex between the HLA antigen and the tumor antigen peptide orderivative thereof, and also provides a pharmaceutical composition fortreating tumors which comprises said CTLs as an active ingredient. Suchcomposition preferably contains physiological saline, phosphate bufferedsaline (PBS), medium, or the like to stably maintain CTLs. It may beadministered, for example, intravenously, subcutaneously, orintradermally. By reintroducing the composition for treating tumorswhich comprises CTLs as an active ingredient into the body of thepatient, the toxic effect of CTLs against the tumor cells is enhanced inSART-3-positive patient and thereby destroys the tumor cells to achievetreatment of the tumor.

Tumor antigen peptides, derivatives thereof, tumor antigen proteins, orrecombinant polypeptide thereof according to the present invention maybe also used as an active ingredient of a diagnostic agent fordiagnosing tumors. Specifically, by using a tumor antigen peptide, orderivative thereof according to the present invention itself as adiagnostic agent to detect the presence of antibodies in a sample (suchas blood or a tumor tissue) obtained from a patient suspected to have atumor, early detection of tumors and diagnosis of recurrence andmetastasis are possible. The same procedure can also be used forselection of tumor patients to whom medicaments comprising as an activeingredient, for example, a tumor antigen peptide of the presentinvention can be applied. In particular, such diagnosis may be conductedusing immunoblotting, RIA, ELISA, or a fluorescent or luminescent assay.

Furthermore, in recent years, a new detection method has beenestablished for detecting antigen-specific CTLs using a complex betweenthe antigen peptide and an HLA antigen (Science, 274:94, 1996). Earlydetection of tumors and diagnosis of reoccurrence or metastasis arepossible by applying a complex between a tumor antigen peptide orderivative thereof according to the present invention and an HLA antigento the above detection method, and thereby detecting tumorantigen-specific CTLs. The same procedure can also be used for selectionof tumor patients to whom a medicine comprising as an active ingredient,for example, a tumor antigen peptide of the present invention can beapplied, or for determination of the therapeutic effect of saidmedicament. Thus, the present invention also provides a diagnostic agentfor tumors comprising a tumor antigen peptide or derivative thereofaccording to the present invention.

In particular, such diagnosis may be conducted by preparing a tetramerof a complex between an HLA antigen fluorescently labeled according tothe method described in the literature (Science, 274:94, 1996) and atumor antigen peptide, and using it to quantitatively determine theantigen peptide-specific CTLs in peripheral blood lymphocytes of apatient suspected to have a tumor using a flow cytometer.

The present invention also provides OK-CTL (deposit number FERM BP-6818)that is CTL established from tumor-infiltrating lymphocytes derived fromcolon cancer. OK-CTL has proved to be HLA-A2-restricted. Accordingly,tumor antigen proteins and HLA-A2-restricted tumor antigen peptides maybe newly found by using OK-CTL. For details, see Example 8 below.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is further illustrated by the following examples,but is not limited by these examples in any respect.

Reference 1

Establishment of Cytotoxic T Lymphocyte (CTL) Cell Line AgainstEsophageal Cancer Cell Line

According to the description of Nakao et al., Cancer Res., 55:4248-4252(1995), CTL against esophageal cancer cell line KE-4, which belongs tosquamous cell carcinomas when classified on the basis of tissue type,was established from peripheral blood mononuclear cells of a patient,named KE-4CTL, and used in the following experiments. Esophageal cancercell lines KE-4 and KE-4CTL have been deposited at The NationalInstitute of Bioscience and Human Technology (1-1-3 Higashi, Tsukuba,Ibaraki, Japan) under International Deposition Nos. FERM BP-5955 andFERM BP-5954, respectively, both on May 23, 1997. Further, typing of HLAclass I molecules of KE-4 was conducted according to the above-mentioneddescription of Nakao et al., to find that they are HLA-A2402, -A2601,-B54, -B60, -Cw1, and -Cw3.

Reference 2

Preparation of HLA-A2402 cDNA

According to the description of Nakao et al., Cancer Res., 55: 4248-4252(1995), a recombinant plasmid was prepared from KE-4 by incorporatingcDNA for HLA-A2402 (Genbank Accession No.M64740) into an expressionvector pCR3 (INVITROGEN).

Reference 3

Preparation of cDNA Library Derived from KE-4

Poly (A)⁺ mRNA was prepared from KE-4 by isolation of total RNA fractionand purification on oligo (dT) column using mRNA Purification system(Pharmacia Biotech) according to the manufacturer's protocol. cDNAshaving Not I adapter and Sca I adapter linked to each terminus wereprepared from the mRNAs using SuperScript Plasmid System (GIBCO BRL)according to the manufacturer's protocol, and then ligated into therestriction sites Not I and Sal I of an expression vector, plasmidpSV-SPORT1 (GIBCO BRL), to yield recombinant plasmids. The recombinantplasmids were introduced into E. coli. ElectroMAX DH10B™ cells (GIBCOBRL) using electric pulses in Gene Pulser (Bio-Rad) under a condition of25 μF and 2.5 kV. Transformants into which the recombinant plasmids hadbeen introduced were selected in LB medium (1% bacto-trypton, 0.5% yeastextract, 0.5% NaCl, pH7.3) containing ampicillin (50 μg/ml).

EXAMPLE 1

Screening of Novel Tumor Antigen Protein Gene

The recombinant plasmid DNAs were recovered as follows, from pools ofabout 100 transformants described in Reference 3. A hundredtransformants were introduced and cultured in each well of 96-wellU-bottomed microplate containing LB medium plus ampicillin (50 μg/ml).Part of the culture was then transferred to another 96-well U-bottomedmicroplate containing 0.25 ml of TYGPN medium per well (F. M. Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.),and cultured at 37° C. for 48 hours. The remaining cultures in LB mediumon the microplate were stored in frozen. Preparation of recombinantplasmid DNAs from transformants cultured in TYGPN medium was achieved inthe microplate by the alkaline lysis method (F. M. Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Therecombinant plasmid DNAs recovered by isopropanol precipitation weresuspended in 50 μl of 10 mM Tris, 1 mM EDTA, pH 7.4, containing 20 ng/mlRNase.

The recombinant plasmid for KE-4 cDNA and the recombinant plasmid forHLA-A2402 cDNA were doubly transfected into fibroblast cell line VA-13cells (RIKEN CELL BANK, The Institute of Physical and Chemical Research;Ann. Med. Exp. Biol. Fenn., 44:242-254, 1966) using the Lipofectinmethod as follows. Seven thousands VA-13 cells were placed into eachwell of 96-well flat-bottomed microplate, and incubated for 2 days in100 μl of RPMI 1640 medium containing 10% FCS. Using Lipofectin reagent(GIBCO BRL), a 30 μl portion of mixture 70 μl consisting of 25 μl of therecombinant plasmid for KE-4 cDNA corresponding to about 100transformants, 10 μl (200 ng) of the recombinant plasmid for HLA-A2402cDNA described in Reference 2, and 35 μl of about 35-fold dilutedLipofectin reagent was added to VA-13 cells, and allowed to doublytransfect them. Transfectants were prepared in duplicate. After 5 hours,the transfectants was added with 200 μl of culture medium containing 10%FCS, and further incubated at 37° C. for 72 hours. After removing theculture medium, 10,000 KE-4CTL cells were added to each well, andcultured at 37° C. for 24 hours in 100 μl of culture medium containing10% FCS and 25 U/ml IL-2. The culture medium was recovered, and theamount of IFN-γ in the culture was measured by ELISA as described below.

Specifically, an anti-human IFN-γ mouse monoclonal antibody was adsorbedon wells of 96-well microplate as a solid-phased antibody, and afterblocking non-specific bindings with bovine serum albumin, the antibodywas allowed to bind to IFN-γ in the above-described sample. Anti-humanIFN-γ rabbit polyclonal antibody as a detection antibody was thenallowed to bind, and after binding to an anti-rabbit immunoglobulin goatantibody labeled with alkaline phosphatase, para-nitrophenyl phosphatewas reacted as a chromogenic substrate. After quenching the reaction byadding an equal volume of 1N NaOH, absorbance at 405 nm was measured.The absorbance was compared with that obtained with standard IFN-γ todetermine the amount of IFN-γ in the sample.

Regarding the groups in which high production of IFN-γ was observed, thecorresponding frozen-stored pools of about 100 transformants containingrecombinant plasmids for KE-4 cDNA were used in the following screening.The pools of the transformants were plated on LB agar medium containingampicillin (50 μg/ml) to obtain colonies. Two hundreds colonies for eachgroup were cultured as described above so that a single kind oftransformant is included in each well, thereby preparing recombinantplasmid DNAs for KE-4 cDNA. Then, VA-13 cells were doubly transfectedwith the recombinant plasmid for KE-4 cDNA and the recombinant plasmidfor HLA-A2402 cDNA, followed by co-cultivation with KE-4CTL, and IFN-γproduced due to KE-4CTL reaction was quantitatively determined asdescribed above so as to select positive plasmids. In this manner, asingle KE-4 cDNA recombinant plasmid clone was selected and named clone13. Additional analysis revealed that clone 13 was incorporated withabout 1.2 kb cDNA. Furthermore, similar procedures were repeated withclone 13 to determine the amount of IFN-γ produced by KE-4CTL accordingto a similar method to that described above. The results are shown inTable 2. TABLE 2 Amount of IFN-γ Target cell produced by KE-4CTL (pg/ml)VA-13 + HLA-A2402 326 VA-13 + HLA-A2402 + clone 13 775

When compared to VA-13 transfected with only HLA-A2402, KE-4CTL reactedmore strongly to VA-13 doubly transfected with HLA-A2402 and clone 13,and produced more IFN-γ. This result indicated that the protein encodedby clone 13 is a tumor antigen protein.

EXAMPLE 2

Cloning of Full-Length cDNA Clone Encoding Tumor Antigen Protein

In order to determine the length of the full-length cDNA geneincorporated in clone 13 obtained in Example 1, Northern Hybridizationwas conducted as described blow.

First of all, RNAs were prepared from esophageal cancer cell line KE-4using RNAzol B (TEL-TEST, Inc.). Five μg of RNA was denatured in thepresence of formamide and formaldehyde, electrophoresed on agarose, andthen transferred and fixed onto Hybond-N+ Nylon membrane (Amersham). Theinserted sequence region of clone 13 was labeled with ³²P usingMultiprime DNA labeling system (Amersham) to prepare a DNA probe.According to the known method (Nakayama et al., Bio-Jikken-Illustrated,vol. 2, “Idenshi-Kaiseki-No-Kiso (A Basis for Gene Analysis)”, pp.148-151, Shujunsha, 1995), this probe was allowed to hybridize to RNAson the membranes, and subjected to autoradiography to detect mRNA forcDNA incorporated in clone 13, indicating that the mRNA was about 3.8 kbin full length. Then, the full-length cDNA clone containing clone 13 asprepared above was cloned. KE-4-derived cDNA Library described inReference 3 was plated on LB agar medium containing ampicillin (50μg/ml) to obtain colonies. The colonies were then transferred to andfixed on Hybond-N+ nylon membrane (Amersham) according to themanufacturer's protocol. DNA probe in which the insertion sequence ofclone 13 was labeled with ³²P was employed for hybridization andautoradiography under similar conditions to those mentioned above inorder to select colonies representing positive transformants.Recombinant plasmids were then recovered from the many coloniesselected, treated with restriction enzymes Not I and Sal I, and thenelectrophoresed on agarose to determine the length of incorporatedcDNAs. A recombinant plasmid incorporating cDNA of about 3.8 kb wasselected, and named clone K. VA-13 Cells were then doubly transfectedwith the recombinant plasmid clone K incorporating cDNA for the tumorantigen protein gene and another recombinant plasmid containing cDNA forHLA-A2402 as described above, and the cells were used as target cells.The amount of IFN-γ produced by the reaction of KE-4CTL was determinedaccording to the method as described above. The results are shown inTable 3. TABLE 3 Amount of IFN-γ Target cell produced by KE-4CTL (pg/ml)VA-13 + HLA-A2403 342 VA-13 + HLA-A2402 + clone K 627

When compared to VA-13 transfected with only HLA-A2402, KE-4CTL reactedmore strongly to VA-13 doubly transfected with HLA-A2402 and clone K,and produced more IFN-γ. This result indicated that the protein encodedby clone K is a tumor antigen protein. The tumor antigen protein encodedby clone K is named SART-3 (squamous cell carcinoma antigens recognizedby T cells-3).

EXAMPLE 3

Determination of Base Sequence of Tumor Antigen Protein Gene

The base sequence of the DNA of tumor antigen protein SART-3 as obtainedin Example 3 was determined using DyeDeoxy Terminator Cycle Sequencingkit (Perkin-Elmer). The base sequence thus determined is shown in SEQ IDNO: 2. The full-length of the cDNA was 3798 base pairs. Amino acidsequence (963 amino acids) encoded by the base sequence of SEQ ID NO: 2is shown in SEQ ID NO: 1. Comparison of the base sequence shown in SEQID NO: 2 to known sequences using GenBank data base revealed that thebase sequence of tumor antigen protein SART-3 has a novel base sequencethat is different from gene KIAA0156 registered at GenBank underAccession No. D63879 in terms of a single base (at position 108 ofKIAA0156), which function has not been demonstrated.

EXAMPLE 4

Selection of Candidate Peptides

There are certain rules (motifs) in the sequences of antigen peptidesthat should be bound and presented by HLA antigens. Regarding the motiffor HLA-A24, it is known that in the sequence of peptides consisting of8 to 11 amino acids, the amino acid at position 2 is tyrosine,phenylalanine, methionine, or tryptophan, and the amino acid at theC-terminus is phenylalanine, tryptophan, leucine, isoleucine, ormethionine (Immunogenetics, 41:178, 1995; J. Immunol., 152:3913, 1994;J. Immunol., 155:4307, 1994). According to the motifs, peptide portionsconsisting of 8 to 11 peptides having the above motifs were selectedfrom the amino acid sequence of tumor antigen protein SART-3 shown inSEQ ID NO: 1. Those examples of the selected peptides are shown in SEQID NOs: 3-24. These peptides were synthesized at Biologica Co. by theFmoc method.

Then, 1.8×10⁴ VA-13 cells were transfected with a recombinant plasmid ofHLA-A2402 cDNA by the Lipofectin method to express HLA-A2402 accordingto the literature (J. Exp. Med., 187:277, 1998). To these cells, variouspeptides having a binding motif for HLA-A24 that had precedentlysynthesized were each added at 10 μM over two hours in order to pulsethe cells. The cells were then cultured with 2×10⁴ KE-4CTLs for 18hours, and the amount of IFN-γ produced by KE-4CTL in the culturesupernatant was determined by the ELISA method. The results of thisdetermination are shown in Table 4, which performed on seven peptides,that is, a peptide “109-118” comprising the sequence from position 109to position 118 (SEQ ID NO: 3), a peptide “172-181” comprising thesequence from position 172 to position 181 (SEQ ID NO: 4), a peptide“284-292” comprising the sequence from position 284 to position 292 (SEQID NO: 5), a peptide “315-323” comprising the sequence from position 315to position 323 (SEQ ID NO: 6), a peptide “416-425” comprising thesequence from position 416 to position 425 (SEQ ID NO: 7), a peptide“426-434” comprising the sequence from position 426 to position 434 (SEQID NO: 8), and a peptide “448-456” comprising the sequence from position448 to position 456 (SEQ ID NO: 9), in the amino acid sequence of tumorantigen protein SART-3. TABLE 4 Peptides IFN-γ in the supernatant(pg/ml) “109-118” 928 “172-181” 830 “284-292” 794 “315-323” 880“416-425” 731 “426-434” 833 “448-456” 754 None 677

When compared to cells pulsed with no peptide, KE-4CTLs reacted morestrongly to cells pulsed with the peptides, and produced more IFN-γ.This result indicated that the seven peptides function as tumor antigenpeptides.

EXAMPLE 5

Synthesis of Tumor Antigen Peptides

The seven peptides described above were synthesized by the solid phasemethod as shown below.

(1) Synthesis of SART-3 “109-118”Val-Tyr-Asp-Tyr-Asn-Cys-His-Val-Asp-Leu (SEQ ID NO: 3)

Fmoc-Leu-Alko Resin (0.55 mmol/g, 100-200 mesh) was used as a resin.Using 100 mg of this resin, the synthesis was started according toSchedule 1 described below to couple the following residues in order:Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-His(Boc)-OH, Fmoc-Cys(Trt)-OH,Fmoc-Asn-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, andFmoc-Val-OH. After the coupling, the procedures were conducted up toStep 3 of Schedule 1 to obtain a peptide resin.

To this peptide resin, 2 ml of Reagent K (the solution of 5% phenol, 5%thioanisole, 5% H₂O, and 2.5% ethanedithiol in TFA) was added and themixture was allowed to react for 2.5 hours at room temperature. Whilecooling with ice, 10 ml of diethyl ether was added to the reaction, themixture was stirred for 10 minutes, filtered, and washed with 10 ml ofdiethyl ether. To the filter cake, 10 ml of aqueous acetic acid wasadded, and the mixture was stirred for 30 minutes. The resin was thenfiltered, and washed with 4 ml of aqueous acetic acid. Afterlyophilizing the filtrate and the wash, the crude peptide obtained wasdissolved in aqueous acetic acid, and injected into a reverse phasepacking material, YMC-PACK ODS-A column (30φ×250 mm) that had beenpre-equilibrated with 0.1% aqueous TFA. The column was washed with 0.1%aqueous TFA, and elution at a flow rate of 7 ml/min was then conducted,while increasing the concentration of acetonitrile up to 25% over 180minutes. The eluate was monitored by A 220 nm. The fractions containingthe desired product were combined together and lyophilized to obtain31.0 mg of Val-Tyr-Asp-Tyr-Asn-Cys-His-Val-Asp-Leu.

The peptide obtained, Val-Tyr-Asp-Tyr-Asn-Cys-His-Val-Asp-Leu, had aretention time of 19.3 minutes in an analysis using a reverse phasepacking material, YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 16 to 46% containing0.1% TFA, and the results of amino acid analysis (Cys being notdetected) and mass spectrometry of the product were consistent with thetheoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 8 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Asx: 2.77 (3)

Val: 1.70 (2)

*Leu: 1.00 (1)

Tyr: 1.98 (2)

His: 0.91 (1)

Mass spectrum (FAB) [M+H]⁺: 1241 TABLE 5 Schedule 1 Duration (min) ×Steps the number of treatments 1. (washing) DMF 1.2 ml 1 × 2 2.(deprotection) 50% piperidine/DMF 12 × 1  3. (washing) DMF 1.2 ml 1 × 74. (coupling) each amino-protected amino 30 × 1  acid (5equivalents)/NMP solution 0.9 ml, DIC (5 equivalents)/NMP solution 0.3ml 5. (washing) DMF 1.2 ml 1 × 2 6. (coupling) each amino-protectedamino 30 × 1  acid (5 equivalents)/NMP solution 0.9 ml, DIC (5equivalents)/NMP solution 0.3 ml 7. (washing) DMF 1.2 ml 1 × 4(2) Synthesis of SART-3 “172-181”Leu-Phe-Glu-Lys-Ala-Val-Lys-Asp-Tyr-Ile (SEQ ID NO: 4)

According to a similar manner to that described in above (1), using 100mg of Fmoc-Ile-Alko Resin (0.41 mmol/g, 100-200 mesh), Fmoc-Tyr(tBu)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Ala-OH,Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, and Fmoc-Leu-OH werecoupled in order, and the product was then deprotected. The crudepeptide obtained was dissolved in aqueous acetic acid and injected intoa reverse phase packing material YMC-PACK ODS-A column (30φ×250 mm) thathas been pre-equilibrated with 0.1% aqueous TFA. The column was washedwith 0.1% aqueous TFA, and the elution at a flow rate of 7 ml/min wasthen conducted, while increasing the concentration of acetonitrile up to30% over 300 minutes. The eluate was monitored by A 220 nm. Thefractions containing the desired product were combined together andlyophilized to obtain 66.3 mg ofLeu-Phe-Glu-Lys-Ala-Val-Lys-Asp-Tyr-Ile.

The peptide obtained, Leu-Phe-Glu-Lys-Ala-Val-Lys-Asp-Tyr-Ile, had aretention time of 23.8 minutes in an analysis using a reverse phasepacking material YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 12 to 42% containing0.1% TFA, and the results of amino acid analysis and mass spectrometryof the product were consistent with the theoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 12 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Asx: 0.94 (1)

Glx: 1.03 (1)

Ala: 1.00 (1)

Val: 0.88 (1)

Ile: 0.92 (1)

*Leu: 1.00 (1)

Tyr: 0.96 (1)

Phe: 0.97 (1)

Lys: 1.45 (2)

Mass spectrum (FAB): [M+H]⁺: 1225

(3) Synthesis of SART-3 “284-292” Asn-Tyr-Asn-Lys-Ala-Leu-Gln-Gln-Leu(SEQ ID NO: 5)

According to a similar manner to that described in above (1), using 100mg of Fmoc-Leu-Alko Resin, Fmoc-Gln-OH, Fmoc-Gln-OH, Fmoc-Leu-OH,Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn-OH, Fmoc-Tyr(tBu)-OH, andFmoc-Asn-OH were coupled in order, and the product was then deprotected.The crude peptide obtained was dissolved in aqueous acetic acid andinjected into a reverse phase packing material YMC-PACK ODS-A column(30φ×250 mm) that has been pre-equilibrated with 0.1% aqueous TFA. Thecolumn was washed with 0.1% aqueous TFA, and the elution at a flow rateof 7 ml/min was then conducted, while increasing the concentration ofacetonitrile up to 30% over 300 minutes. The eluate was monitored by A220 nm. The fractions containing the desired product were combinedtogether and lyophilized to obtain 25.0 mg ofAsn-Tyr-Asn-Lys-Ala-Leu-Gln-Gln-Leu.

The peptide obtained, Asn-Tyr-Asn-Lys-Ala-Leu-Gln-Gln-Leu, had aretention time of 19.0 minutes in an analysis using a reverse phasepacking material YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 12 to 42% containing0.1% TFA, and the results of amino acid analysis and mass spectrometryof the product were consistent with the theoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 12 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Asx: 1.87 (2)

Glx: 2.03 (2)

Ala: 0.98 (1)

*Leu: 2.00 (2)

Tyr: 0.99 (1)

Lys: 0.97 (1)

Mass spectrum (FAB): [M+H]⁺: 1091

(4) Synthesis of SART-3 “315-323” Ala-Tyr-Ile-Asp-Phe-Glu-Met-Lys-Ile(SEQ ID NO: 6)

According to a similar manner to that described in above (1), using 100mg of Fmoc-Ile-Alko Resin (0.62 mmol/g, 100-200 mesh), Fmoc-Lys(Boc)-OH,Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Ala-OH were coupled in order,and the product was then deprotected. The crude peptide obtained wasdissolved in aqueous acetic acid and injected into a reverse phasepacking material YMC-PACK ODS-A column (30φ×250 mm) that has beenpre-equilibrated with 0.1% aqueous TFA. The column was washed with 0.1%aqueous TFA, and the elution at a flow rate of 7 ml/min was thenconducted, while increasing the concentration of acetonitrile up to 40%over 180 minutes. The eluate was monitored by A 220 nm. The fractionscontaining the desired product were combined together and lyophilized toobtain 15.4 mg of Ala-Tyr-Ile-Asp-Phe-Glu-Met-Lys-Ile.

The peptide obtained, Ala-Tyr-Ile-Asp-Phe-Glu-Met-Lys-Ile, had aretention time of 19.6 minutes in an analysis using a reverse phasepacking material YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 21 to 51% containing0.1% TFA, and the results of amino acid analysis (Met being notdetected) and mass spectrometry of the product were consistent with thetheoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 12 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Asx: 0.91 (1)

Glx: 1.06 (1)

Ala: 1.06 (1)

Ile: 1.69 (2)

Tyr: 0.81 (1)

*Phe: 1.00 (1)

Lys: 0.87 (1)

Mass spectrum (FAB): [M+H]⁺: 1130

(5) Synthesis of SART-3 “416-425”Asp-Tyr-Val-Glu-Ile-Tyr-Gln-Ala-Tyr-Leu (SEQ ID NO: 7)

According to a similar manner to that described in above (1), using 100mg of Fmoc-Leu-Alko Resin, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gln-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH,Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH were coupled in order, and theproduct was then deprotected. The crude peptide obtained was dissolvedin aqueous acetic acid and injected into a reverse phase packingmaterial YMC-PACK ODS-A column (30φ×250 mm) that has beenpre-equilibrated with 0.1% aqueous TFA. The column was washed with 0.1%aqueous TFA, and the elution at a flow rate of 7 ml/min was thenconducted, while increasing the concentration of acetonitrile up to 35%over 180 minutes. The eluate was monitored by A 220 nm. The fractionscontaining the desired product were combined together and lyophilized toobtain 18.9 mg of Asp-Tyr-Val-Glu-Ile-Trp-Gln-Ala-Tyr-Leu.

The peptide obtained, Asp-Tyr-Val-Glu-Ile-Trp-Gln-Ala-Tyr-Leu, had aretention time of 20.5 minutes in an analysis using a reverse phasepacking material YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 25 to 55% containing0.1% TFA, and the results of amino acid analysis (Trp being notdetected) and mass spectrometry of the product were consistent with thetheoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 10 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Asx: 1.00 (1)

Glx: 2.09 (2)

Ala: 1.04 (1)

Val: 0.89 (1)

Ile: 0.86 (1)

*Leu: 1.00 (1)

Tyr: 1.95 (2)

Mass spectrum (FAB): [M+H]⁺: 1300

(6) Synthesis of SART-3 “426-434” Asp-Tyr-Leu-Arg-Arg-Arg-Val-Asp-Phe(SEQ ID NO: 8)

According to a similar manner to that described in above (1), using 100mg of Fmoc-Phe-Alko Resin (0.72 mmol/g, 100-200 mesh),Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH,Fmoc-Arg(Pmc)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OHwere coupled in order, and the product was then deprotected. The crudepeptide obtained was dissolved in aqueous acetic acid and injected intoa reverse phase packing material YMC-PACK ODS-A column (30φ×250 mm) thathas been pre-equilibrated with 0.1% aqueous TFA. The column was washedwith 0.1% aqueous TFA, and the elution at a flow rate of 7 ml/min wasthen conducted, while increasing the concentration of acetonitrile up to25% over 240 minutes. The eluate was monitored by A 220 nm. Thefractions containing the desired product were combined together andlyophilized to obtain 34.0 mg of Asp-Tyr-Leu-Arg-Arg-Arg-Val-Asp-Phe.

The peptide obtained, Asp-Tyr-Leu-Arg-Arg-Arg-Val-Asp-Phe, had aretention time of 20.1 minutes in an analysis using a reverse phasepacking material YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 12 to 42% containing0.1% TFA, and the results of amino acid analysis and mass spectrometryof the product were consistent with the theoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 12 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Asx: 1.90 (2)

Val: 0.95 (1)

*Leu: 1.00 (1)

Tyr: 1.00 (1)

Phe: 0.99 (1)

Arg: 2.93 (3)

Mass spectrum (FAB): [M+H]⁺: 1239

(7) Synthesis of SART-3 “448-456” Ala-Phe-Thr-Arg-Ala-Leu-Glu-Tyr-Leu(SEQ ID NO: 9)

According to a similar manner to that described in above (1), using 100mg of Fmoc-Leu-Alko Resin, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Thr(tBu)-OH,Fmoc-Phe-OH, and Fmoc-Ala-OH were coupled in order, and the product wasthen deprotected. The crude peptide obtained was dissolved in aqueousacetic acid and injected into a reverse phase packing material YMC-PACKODS-A column (30φ×250 mm) that has been pre-equilibrated with 0.1%aqueous TFA. The column was washed with 0.1% aqueous TFA, and theelution at a flow rate of 7 ml/min was then conducted, while increasingthe concentration of acetonitrile up to 30% over 240 minutes. The eluatewas monitored by A 220 nm. The fractions containing the desired productwere combined together and lyophilized to obtain 22.8 mg ofAla-Phe-Thr-Arg-Ala-Leu-Glu-Tyr-Leu.

The peptide obtained, Ala-Phe-Thr-Arg-Ala-Leu-Glu-Tyr-Leu, had aretention time of 18.1 minutes in an analysis using a reverse phasepacking material YMC-PACK ODS-AM column (4.6φ×250 mm) eluted with alinear gradient of acetonitrile concentration from 20 to 50% containing0.1% TFA, and the results of amino acid analysis and mass spectrometryof the product were consistent with the theoretical values.

Amino Acid Analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 12 hours;

Analysis method: the ninhydrin method;

*Reference amino acid; Theoretical values are indicated in parentheses:

Thr: 0.91 (1)

Glx: 1.03 (1)

Ala: 1.91 (2)

*Leu: 2.00 (2)

Tyr: 1.00 (1)

Phe: 0.97 (1)

Arg: 0.97 (1)

Mass spectrum (FAB): [M+H]⁺: 1083

EXAMPLE 6

CTL Induction from Peripheral Blood Lymphocytes by Tumor AntigenPeptides and Derivatives Thereof.

The peptides “109-118” (SEQ ID NO: 3) and “315-323” (SEQ ID NO: 6)synthesized as shown in Example 5 were investigated for their ability toinduce antigen-specific CTLs from peripheral blood lymphocytes.

Using the Ficoll method, lymphocytes were separated from peripheralblood of healthy donors who were heterozygous for A24 in the HLA-A locus(referred to as HD1 and HD2, respectively). The lymphocytes were placedinto wells of a 24-well plate at 2×10⁶ cells/well, and cultured in thelymphocyte medium. The above tumor antigen peptides were added to theculture medium at 10 μM to stimulate the peripheral blood lymphocytes.After one week, the above tumor antigen peptide was added to attain 10μM together with about 2×10⁵ cells of X-radiated (50 Gy) peripheralblood lymphocytes for the second stimulation. After additional one week,the third stimulation was conducted in a similar manner. Culturedlymphocytes were harvested one week after the third stimulation. Usingas target cells (1×10⁴ cells) MT-2, which is an HLA-A2402-positiveleukemia T cell line expressing SART-3, and RPMI8402, which is anHLA-A2402-negative leukemia T cell line expressing SART-3, the amount ofIFN-γ in the culture medium produced by the above lymphocytes (8×10⁴cells) in response to the target cells was measured in accordance with asimilar ELISA method to that in Example 1. The results are shown inTable 6. TABLE 6 IFN-γ in Supernatant (pg/ml) HD1 HD2 Antigen PeptidesMT-2 RPMI8402 MT-2 RPMI8402 “109-118” 1771 159 2078 28 “315-323” 2041 26974 40 None 552 154 413 69

Peripheral blood lymphocytes stimulated with “109-118” and “315-323”peptides reacted to MT-2 (HLA-A24-positive) but not to RPMI8402(HLA-A24-negative), indicating that CTLs specific for tumor antigenpeptide were induced in a HLA-A24-restricted manner.

Likewise, a similar experiment can be conducted using COS-7 cells (ATCCNo. CRL1651) or VA-13 cells (RIKEN CELL BANK, The Institute of Physicaland Chemical Research) into which an expression plasmid for HLA-A24 cDNAhas been introduced and which have been pulsed with the above peptides,instead of MT-2 used in the present experiment (J. Exp. Med., 187:277,1998).

EXAMPLE 7

Establishment of Cytotoxic T Lymphocytes (CTLs) Cell Line fromTumor-Infiltrating Lymphocytes (TILs) derived from Colon Cancer

TILs from a surgical sample taken from a patient with sigmoid coloncancer (HLA-A0207-positive) were cultured on 24-well plate in a culturemedium consisting of 45% RPMI, 45% AIM-V (GIBCO BRL), and 10% FCSsupplemented with 100 U/ml interleukin-2 and 0.1 mM NEAA (GIBCO BRL)(hereinafter referred to as lymphocyte medium). During the first twodays of the cultivation, an anti-CD3 antibody NU-T3 (NichireiCorporation) was added to the culture medium at 1 μg/ml. The cultivationwas continued for more than 30 days, and a CTL line that is restrictedto HLA-A2 was established, the CTL line being named OK-CTL. OK-CTL wasdeposited at The National Institute of Bioscience and Human Technology,Agency of Industrial Science and Technology (1-1-3 Higashi, Tsukuba,Ibaraki, Japan) (designation of microorganism: OK-CTL; deposition date:Aug. 3, 1999; deposit number: FERM BP-6818).

According to the description in Nakao et al., Cancer Res., 55:4248-4252(1995), recombinant plasmids were prepared from SW620 cells (ATCC No.CCL-227), in which cDNAs for HLA-A0201 (GenBank Accession No. M84379)was incorporated into an expression vector pCR3 (INVITROGEN). Using, astarget cells, transfectants that had been prepared by doublytransfecting a cell line derived from African green monkey kidney, COS-7(ATCC No. CRL1651) (1×10⁴ cells) with recombinant plasmid clone Kincorporated with the SART-3 gene cDNA, and with a recombinant plasmidincorporated with HLA-A0201 cDNA using a Lipofectin method similar to inExample 1, the amount of IFN-γ produced by 5×10⁴ OK-CTLs in response tothe target cells was measured by ELISA. As control groups, anon-treatment group wherein no plasmid was transfected, and a groupwherein recombinant plasmid clone K and the recombinant plasmidincorporated with HLA-A2402 cDNA were doubly transfected were designed.The result is shown in Table 7. TABLE 7 Amount of IFN-γ Target cellsProduced by OK-CTL (pg/ml) COS-7 653 COS-7 + HLA-A0201 + K 2401 COS-7 +HLA-A2402 + K 600

OK-CTL reacted more strongly to the target cells doubly transfected withrecombinant plasmid clone K incorporated with SART-3 gene cDNA and withthe recombinant plasmid incorporated with HLA-A0201 cDNA, and producedmore IFN-γ, compared to other target cell groups. This result indicatesthat the antigen peptides of tumor antigen protein SART-3 is presentedon HLA-A0201, and OK-CTL recognizes it, suggesting that SART-3 containsHLA-A2-restricted tumor antigen peptides.

EXAMPLE 8

Identification of HLA-A2-Restricted Tumor Antigen Peptides

On the basis of the amino acid sequence of tumor antigen protein SART-3shown in SEQ ID NO: 1, peptide sequences consisting of nine or ten aminoacid residues that were expected to be capable of binding to HLA-A0201were searched on internet using a software of NIH BIMAS(http://bimas.dcrt.nih.gov/molbio/hla_bind/). Those examples of thesearched peptides are shown in SEQ ID NOs: 25-52. These peptides weresynthesized at Biologica Co. by the Fmoc method.

Then, 1×10⁴ T2 cells, T-B hybridoma cell line that is HLA-A0201 positiveand that lacks in an capability to present endogenous peptides werepulsed with each of peptides expected to be capable of binding toHLA-A0201 that had precedently synthesized at 10 μM over two hours. Thecells were then cultured with 6×10⁴ OK-CTLs for 18 hours, and the amountof IFN-γ produced by OK-CTL in the culture supernatant was determined byELISA. The results of this determination are shown in Table 8, whichperformed on five peptides, that is, a peptide “152-160” comprising thesequence from position 152 to position 160 (SEQ ID NO: 25), a peptide“249-257” comprising the sequence from position 249 to position 257 (SEQID NO: 26), a peptide “302-310” comprising the sequence from position302 to position 310 (SEQ ID NO: 27), a peptide “309-317” comprising thesequence from position 309 to position 317 (SEQ ID NO: 28), and apeptide “386-394” comprising the sequence from position 386 to position394 (SEQ ID NO: 29), in the amino acid sequence of tumor antigen proteinSART-3. TABLE 8 Peptides IFN-γ in the supernatant (pg/ml)* “152-160” 162“249-257” 209 “302-310” 190 “309-317” 231 “386-394” 122*The values are those subtracted by the amount of produced IFN-γ by T2cells pulsed with no peptide.

KE-4CTLs reacted more strongly to cells pulsed with the peptides, andproduced more IFN-γ, compared to cells pulsed with no peptide. Thisresult indicates that the five peptides function as HLA-A2-restrictedtumor antigen peptides.

Likewise, a similar experiment can be conducted using COS-7 cells (ATCCNo. CRL1651) or VA-13 cells (RIKEN CELL BANK, The Institute of Physicaland Chemical Research) into which an expression plasmid for HLA-A0201cDNA has been introduced instead of the T2 cells as used in the presentexperiment (J. Exp. Med., 187:277, 1998).

Sequence Listing Free Text

In the amino acid sequence shown in SEQ ID NO: 53, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the tenthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 54, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the tenthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 55, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the ninthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 56, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the ninthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 57, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the tenthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 58, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the ninthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 59, the second amino acidis phenylalanine, tyrosine, methionine, or tryptophan, and the ninthamino acid is phenylalanine, leucine, isoleucine, tryptophan, ormethionine.

In the amino acid sequence shown in SEQ ID NO: 60, the second amino acidis leucine, methionine, valine, isoleucine, or glutamine, and the ninthamino acid is valine, or leucine.

In the amino acid sequence shown in SEQ ID NO: 61, the second amino acidis leucine, methionine, valine, isoleucine, or glutamine, and the ninthamino acid is valine, or leucine.

In the amino acid sequence shown in SEQ ID NO: 62, the second amino acidis leucine, methionine, valine, isoleucine, or glutamine, and the ninthamino acid is valine, or leucine.

In the amino acid sequence shown in SEQ ID NO: 63, the second amino acidis leucine, methionine, valine, isoleucine, or glutamine, and the ninthamino acid is valine, or leucine.

In the amino acid sequence shown in SEQ ID NO: 64, the second amino acidis leucine, methionine, valine, isoleucine, or glutamine, and the ninthamino acid is valine, or leucine.

INDUSTRIAL APPLICABILITY

According to the present invention, a novel tumor antigen protein andgene therefor, tumor antigen peptides derived from said tumor antigenprotein, and derivatives thereof, as well as medicaments, prophylactics,or diagnostics for tumors using such tumor antigen protein, gene, tumorantigen peptides, or derivatives thereof in vivo or in vitro, can beprovided.

1. A method for treating tumors, which comprises administering a tumorantigen protein selected from the group consisting of: (a) a proteincomprising the amino acid sequence of SEQ ID NO: 2, and (b) a proteinthat is encoded by a polynucleotide which hybridizes with apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 understringent hybridization conditions comprising 6×SSC, 50% formamide, and0.5% SDS and a temperature of 42° C., wherein said protein of (a) and(b) give rise to tumor antigen peptide(s) that bind(s) to an HLA antigenand are recognized by cytotoxic T lymphocytes.
 2. A tumor antigenpeptide that is a partial peptide of a protein consisting of the aminoacid sequence of SEQ ID NO: 2, and that binds to an HLA antigen and isrecognized by cytotoxic T lymphocytes.
 3. The tumor antigen peptide ofclaim 2 wherein the HLA antigen is HLA-A24 or HLA-A2.
 4. The tumorantigen peptide of claim 3, which comprises an amino acid sequence shownin any one of SEQ ID NOs: 3-52.
 5. The tumor antigen peptide of claim 4which comprises an amino acid sequence shown in any one of SEQ ID NOs:3-9 and 25-29.
 6. A derivative of the tumor antigen peptide of claim 4,in which the amino acid residue at position 2 and/or the C-terminus inthe amino acid sequence shown in any one of SEQ ID NOs: 3-52 issubstituted by another amino acid residue, and in which the derivativebinds to an HLA antigen and is recognized by cytotoxic T lymphocytes. 7.A derivative of the tumor antigen peptide of claim 5, in which the aminoacid residue at position 2 and/or the C-terminus in the amino acidsequence shown in any one of SEQ ID NOs: 3-9 and 25-29 is substituted byanother amino acid residue, and in which the derivative binds to an HLAantigen and is recognized by cytotoxic T lymphocytes.
 8. The derivativeof claim 6, in which the amino acid residue at position 2 in the aminoacid sequence shown in any one of SEQ ID NOs: 3-24 is substituted bytyrosine, phenylalanine, methionine, or tryptophan, and/or the aminoacid residue at the C-terminus is substituted by phenylalanine, leucine,isoleucine, tryptophan, or methionine.
 9. The derivative of claim 6, inwhich the amino acid residue at position 2 in the amino acid sequenceshown in any one of SEQ ID NOs: 25-52 is substituted by leucine,methionine, valine, isoleucine, or glutamine, and/or the amino acidresidue at the C-terminus is substituted by valine or leucine.
 10. Thederivative of claim 7, which comprises the amino acid sequence shown inany one SEQ ID NOs: 53-64.
 11. A pharmaceutical composition for treatingtumors, which comprises as an active ingredient at least one ofsubstances selected from the tumor antigen peptides and the derivativesaccording to any one of claims 2 to
 10. 12. A recombinant polypeptideobtainable by expressing the recombinant DNA comprising at least one ofDNAs that encode the tumor antigen peptides or the derivatives accordingto any one of claims 2 to
 10. 13. A pharmaceutical composition fortreating tumors, which comprises as an active ingredient the recombinantpolypeptide of claim
 12. 14. A diagnostic agent for tumors, whichcomprises the tumor antigen peptide or the derivative according to anyone of claims 2 to
 10. 15. A diagnostic agent for tumors, whichcomprises the recombinant polypeptide of claim 12.